Cd31 pe clone wm59

To determine cellular origin of circulating B cell activating factor (BAFF) + MVs, flow cytometric analysis was performed on selected PROFLOW cohort samples with an Apogee A60 cytometer (Apogee Flow Systems, Hemel Hempstead, UK). Size-calibrated silica beads (ApogeeMix, Apogee Flow Systems) were used to establish MV gate (Supplemental Fig. S1). Fluorescence gates were created based on Ab isotype controls: PE mouse IgG1κ, FITC mouse IgG1κ, and Alexa Fluor 488 mouse IgG1κ (Supplemental Fig. S2), as well as Abs in DBPS. All Abs were centrifuged 20,000 g for 30 min in 4°C prior application, to remove Ab aggregates. Patient plasma was diluted 1:200 with DPBS, and 200 μL was stained for 30 min in RT with 2 μL of Abs against cellular origin markers and BAFF and directly analyzed. The gating strategy is presented in Supplemental Fig. S3A. CD42b PE and FITC clone HIP1, CD31 PE clone WM59, CD45 FITC clone HI30, CD16 PE and FITC clone 3G8, CD62E PE clone 68-5H11, CD36 PE clone CB38, and CD144 FITC clone 55-7H1 were purchased from BD Biosciences. CD235a FITC clone KC16 was purchased from Beckman Coulter. BAFF Alexa Fluor 488 was purchased from Bio-Techne. BAFF PE clone 1D6 was purchased from Thermo Fisher Scientific.

Flow cytometric analysis of hematopoietic surface markers was analyzed on a FACS Canto II (BD Biosciences, Franklin Lakes, New Jersey, USA) with the following antibodies: CD3-PerCP-Cy5.5 (clone UCHT1), CD19-FITC (clone HIB19), CD31-PE (clone WM59), CD34-APC (clone 581), CD38-PE (clone HIT2), CD43-FITC (clone 1G10), CD14-PE (clone MOP9), CD66b-PE (clone G10F5) (all BD Bioscience), CD45-APC-Cy7 (clone 2D1), CD117-PE-Cy7 (clone 104D2), CD11c-PE-Cy7 (clone 3.9), HLA-DR-FITC (clone LN3), CD235a-Pacific Blue (clone 6A7M) (all eBioscience, San Diego, California, USA), CD33-APC (clone AC104.3E3; Miltenyi Biotec). EBs were dissociated with Accutase (Stemcell Technologies, Vancouver, British Columbia, Canada) for 10–15 min prior to staining with antibodies. Single cells were incubated with 1% human IgG solution (Privigen, CSL Behring, King of Prussia, Pennsylvania, USA) for 30 min at 4 °C to block unspecific binding.
Immunophenotypic analysis of MSCs and iMSCs was performed with CD14-APC (clone M5E2), CD29-PE (clone MAR4), CD31-PE (clone WM59), CD34-APC (clone 581), CD45-APC (clone HI30), CD73-PE (clone AD2), CD90-APC (clone 5E10) (all BD Biosciences), and CD105-FITC (clone MEM-226; ImmunoTools, Friesoythe, Germany). Data was analyzed using the FlowJo software (Tree Star, Ashland, Oregon, USA).

Immunophenotypic surface marker analysis was performed on a FACS Canto II (BD, Heidelberg, Germany) upon staining with CD14 allophycocyanin (APC, clone M5E2; BD), CD29 phycoerythrin (PE, clone MAR4; BD), CD31 PE (clone WM59; BD), CD34 APC (clone 8G12; BD), CD45 APC (clone HI30; BD), CD73 PE (clone AD2; BD), CD90 APC (clone 5E10; BD), CD105 fluorescein isothiocyanate (FITC, clone MEM-226; ImmunoTools, Friesoythe, Germany).